The present invention relates to protein and peptide chemistry. In particular, the present invention relates to the discovery and isolation of novel peptides whose sequences derive from microorganism by-products including Lactobacillus biosurfactants. The invention is also directed to the use of these novel peptides in the inhibition of infections caused by pathogens.
Microorganisms still represent one of the top three causes of death amongst humans and animals. The offending organisms can be bacteria, fungi, protozoa, viruses and other forms, collectively termed as pathogens (or organisms which behave like pathogens under certain situations, such as when the host is immunocompromised). Pathogens use many different factors to cause disease, including adhesins to colonize tissues and surfaces, toxins, slime and other capsular substances, antibiotic-resistance genes, immune modifiers and substances which help escape immune responses, etc.
The primary exogenous mechanism to eradicate offending pathogens is antimicrobial agents, such as antibiotics. However, these are often ineffective due to resistance of pathogenic organisms, inability to eradicate biofilms and poor penetration at the tissue or biomaterial interface.
There are a number of organisms which can infect the host. Of these, S. aureus (particularly methicillin-resistantxe2x80x94MRSA), S. epidermidis (particularly methicillin-resistantxe2x80x94MRSE), Pseudomonas aeruginosa, Enterococcus faecalis(including vancomycin-resistantxe2x80x94VRE) and Bacteroides sp. can be particularly detrimental in wounds and surgical implants. These and other aerobic and anaerobic pathogens cause severe morbidity and death in large patient populations.
Implanted medical devices, such as heart valves and artificial veins and joints, are especially vulnerable to microbial biofilm formation and disease (Gristina, (1987) Science, 237:1588-1595). These surfaces are not well protected by host defenses and such surfaces provide a focal point for infecting pathogens. Closed implants are more frequently associated with life-threatening morbidity. Staphylococcus epidermidis and S. aureus being the major pathogens associated with life-threatening infections (Christensen et al. (1989) xe2x80x9cMicrobial and foreign body factor in the pathogen of medical device infections. In infections associated with in dwelling medical devicesxe2x80x9d, Bisno AL, Waldvogel FA (eds), ASM, Wash., pp. 2759). Thus, various microorganisms share a common ability to form biofilms and colonize human tissues in the presence and absence of foreign bodies.
To combat the significant infectious complication rates associated with the use of surgical implants, several strategies have been employed. Thus far, these have been mainly limited to:improved surgical asepsis techniques, improved regimens of administration of peri-operative systemic antibiotics, local antibiotic irrigation procedures, modification of surface characteristics of surgical implants and impregnation of surgical implants with antibiotics.
Thus, progress has been made, but that based on scientific innovation, has been relatively slow, and most citations in the literature still utilize traditional methods of management, such as use of antibiotics, antiseptics and surgical debridement. Newer approaches to prevent bacteria ascending towards clinical use include vacuum sealed and transparent film dressings, irrigation with antimicrobial agents, use of the port and cap, use of new agents such as deuteroporphyrin against Staphylococcus aureus, gamma interferon, silver sulfadiazone water soluble gel, geomagnetic therapy, and natural remedies such as milliacynic oil and lysozyme. However, few innovations have made a major impact on infection and fatality rates. Indeed, most new approaches involve delivery of an antimicrobial chemical, for example in liposomes or in collagen dressings.
One embodiment of the present invention is directed to microorganism biosurfactant peptides. In a preferred embodiment the microorganism is Lactobacillus. In another preferred embodiment the microorganism is Staphylococcus epidermidis. The present application also contemplates biosurfactant peptides isolated from Streptococcus sp., Bifidobacterium sp. and lactic acid bacteria. In accordance with the present invention, novel peptides derived from or corresponding to by-products produced by Lactobacilli have been isolated and synthesized. These peptides possess anti-microbial properties including the ability to selectively bind to collagen and inhibit infections around wounds and at the site of implants and biofilms which biofilms, for example, are associated with infections in mammals, in vivo. The peptides of the present invention are derived from human microorganism isolates and therefore are non-immunogenic, non-pathogenic, non-carcinogenic and non-invasive when administered to humans.
The present invention also provides methods and compositions for treating infections such as are commonly caused by S. aureus (particularly methicillin-resistantxe2x80x94MRSA), S. epidermidis (particularly methicillin-resistantxe2x80x94MRSE), Pseudomonas aeruginosa, Enterococcus faecalis (including vancomycin-resistantxe2x80x94VRE) and Bacteroides sp., for example.
The present invention also provides methods and compositions for coating devices, including stents, tubes, prosthetics, bandages, intravenous lines, catheters, diaphragms, tampons and the like. The present invention provides compositions containing at least one biosurfactant peptide which inhibits, prevents, treats or reduces the risk of infections in a mammal, and preferably humans.
The present invention also provides a method to identify proteins, peptides, glycoproteins and amino acid sequences from any given microorganism which block the lock and key adherence of pathogens, such as but not limited to Salmonella, Shigella, E. coli, Pseudomonas, Listeria, Yersinia, Enterococcus, Streptococcus and other pathogens including viruses and protozoa.
The biosurfactant peptides of the present invention are at least 5 amino acids in length and possess collagen-receptor binding activity. Collagen is a major extracellular matrix (ECM) component of tissues that has also been shown to be an important site of attachment for invading pathogenic bacteria, like Staphylococcus aureus. 
A preferred biosurfactant peptide of the present invention is a peptide having the sequence Xaa1-Gln-Gly-Xaa2-Val-Ser-Xaa3-Xaa4-Gly-Ser-Cys-Thr-Cys-Ala-Gln-Lys (SEQ ID NO:1), wherein Xaa1 and Xaa2 can be any amino acid.
Preferably, Xaa1, and Xaa2 are Leu. Xaa3 and Xaa4 can either be Phe or Ala. Another preferred biosurfactant peptide of the present invention is a peptide having the sequence Glu-Val-Tyr-Xaa1-Phe-Gln-Glu-Xaa2-Xaa3-Arg (SEQ ID NO:2), wherein Xaa1 can be any amino acid and Xaa2 and Xaa3 can either be Gln or Pro. Another preferred biosurfactant peptide of the present invention is a peptide having the sequence Trp-Asn-Tyr-Val-Ser (SEQ ID NO:3). Still another preferred biosurfactant peptide of the present invention is a peptide having the sequence Arg-Ala-Phe-Ala-Xaa1-Xaa2-Ser-Asn-Trp-Pro-Val-Lys (SEQ ID NO:4), wherein Xaa1 and Xaa2 can be any amino acid. Yet another preferred biosurfactant peptide of the present invention is a peptide having the sequence Gly-Xaa1-Gln-Xaa2-Xaa3-Asp-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Arg (SEQ ID NO: 5), wherein Xaa1, Xaa2, Xaa3 and Xaa4 can be any amino acid and Xaa5, Xaa6, Xaa7, and Xaa8 can be either Ser or His. Homologs and analogs of these peptides are also contemplated by the present invention.
Further, according to the present invention a biosurfactant peptide can be at least 5 amino acids and composed of up to a full length biosurfactant protein, as long as the peptide binds to a collagen receptor and possesses anti-pathogenic activity.
Another aspect of the present invention provides methods of interfering with, blocking or otherwise preventing the interaction or binding of microbial pathogens to collagen receptors on cells, for example, by employing the biosurfactant peptides contemplated by the present invention. As such the present peptides are understood to act as microbial antagonists.
Another aspect of the present invention provides methods of interfering with blocking or otherwise preventing the interaction or binding of microbial pathogens to extracellular matrix substances on cells or on the outer surface conditioning film of a biomaterial, for example.
The present invention also provides compositions for the treatment of infections such as occur at wound sites, implant sites and the like in animals, including humans and includes methods of treating such disorders. The compositions include at least one of the biosurfactant peptides, preferably at least the biosurfactant peptide according to the present invention, is admixed with a pharmaceutically acceptable carrier.
Nucleic acid molecules coding for any of the above biosurfactant peptides of the present invention, expression vectors which include any of such nucleic acid molecules, as well as related host cells containing such nucleotide sequences or vectors, are also contemplated by the present invention.
These and other embodiments of the invention will be readily apparent to those of ordinary skill in view of the disclosure herein.